Compositions of solution for sequencing reaction clean-up

ABSTRACT

Wash solution and method for purifying sequencing reaction product. The wash solution comprises an effective amount of guanidine in a low ionic solution to reduce or eliminate the presence of dye terminators in a sequencing reaction product. In its method aspects, the present invention comprises the addition of the wash solution to the sequencing reaction product prior to filtration, followed by filtration to reduce or eliminate unincorporated dye terminators. The purified sequencing products can then be resuspended and transferred to an appropriate substrate for sequencing or further preparation. Dye blobs formed from unincorporated dye terminators no longer interfere with the electropherograms generated upon electrophoresis of the sample.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Application of InternationalApplication No. PCT/US2003/026557, filed on Aug. 25, 2003, which claimsthe benefit of U.S. Provisional Application No. 60/406,654, filed onAug. 28, 2002.

BACKGROUND

Commercially available DNA sequencing kits, such as the ABI PRISM®BigDye® Terminator v.1.0, 1.1, 2.0, 3.0 and 3.1 Ready Reaction CycleSequencing Kits available from Applied Biosystems, Inc. utilizefluorescently-labeled molecules or dye terminators as a component. Forexample, the dye terminators, deoxynucleoside triphosphates, sequencingenzyme, magnesium chloride and buffer are premixed and are suitable forperforming fluorescence-based cycle sequencing reactions onsingle-stranded or double-stranded DNA templates and on polymerase chainreaction fragments.

Because dye terminators are not natural substrates of DNA polymerase,high concentrations generally must be provided relative to the naturaldNTP substrates to ensure their incorporation into the polymerizingsequencing products. The unincorporated fluorescently-labeled molecules,however, are difficult to remove if present in high concentration. Ifthey are not adequately removed, the unincorporatedfluorescently-labeled molecules can interfere with downstream analysis(e.g., DNA sequencing), such as by co-migrating with short sequencingproducts during electrophoresis. Indeed, these molecules have a tendencyto form insoluble complexes at high concentrations. They areparticularly problematic in reactions that utilize high concentrationsof sequencing chemistries (e.g., 1×, ½× and ¼× strength reactions).

Conventional methods for removing unincorporated dye terminators fromsequencing reactions prior to electrophoresis involve alcoholprecipitation and gel filtration. However, salts compete with sequencingproducts for electrokinetic injection onto capillary sequencinginstruments and also must be removed. Ethanol precipitation has poorsalt removal capabilities which detracts from its utility as a methodfor preparing samples prior to capillary electrophoresis because theefficiency of electrokinetic injection of sequencing products isinversely proportional to the salt concentration. Gel filtration is acentrifuge-based method which is difficult to automate, which isimportant for high throughput DNA sequencing.

Another method of removing unincorporated fluorescently-labeledmolecules such as dye terminators involves using MultiScreen® orMontage™ 384-SEQ ultrafiltration plates commercially available fromMillipore Corporation. These plates are fully automatable and provide acost-and-time efficient alternative to conventional ethanolprecipitation for dye-terminator removal. They operate by vacuumfiltration, thereby eliminating the need for centrifugation, ethanoldrying steps and manifold disassembly routines. Solvents such asformamide or EDTA aid in the solubilization of dye terminators andprevention of aggregate formation. The sequencing products are purifiedby filtering to dryness and then washing salts an dye terminators towaste. The purified sequencing products are then resuspended from themembrane surface and are ready for introduction into a DNA sequencer.

However, the introduction of new sequencing chemistries by variousmanufacturers continues to present purification challenges. In addition,although current ultrafiltration technology provides substantiallypurified DNA sequencing products at reaction strengths of one microliterof sequencing reagent per DNA sequencing reaction (⅛^(th) BDT v3.0), athigher concentrations artifacts known as dye blobs become apparent onelectropherograms. These artifacts are also commonly visible using otherclean-up methods such as gel filtration and alcohol precipitationdespite specific modifications to the protocol recommended by themanufacturer to eliminate them.

It therefore would be desirable to provide a cost-effective andefficient solution for reducing or eliminating unincorporatedfluorescently-labeled molecules and residual salts from sequencingreactions.

SUMMARY

The problems of the prior art have been overcome by the presentinvention, which provides a wash solution and method for purifyingsequencing reaction product. The wash solution preferably comprisesbetween about 1 mM to about 60 mM of guanidine in a low ionic solution.In its method aspects, the present invention comprises the addition ofthe wash solution to the sequencing reaction product prior tofiltration, followed by filtration to reduce or eliminate unincorporateddye terminators. The purified sequencing products can then beresuspended and transferred to an appropriate substrate for sequencingor further preparation. Dye blobs formed from unincorporated dyeterminators no longer interfere with the resolution of electropherogramsgenerated upon electrophoresis of the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an electropherogram of a full scale reaction in accordancewith the prior art;

FIG. 1B is a raw trace of the reaction dye blobs of FIG. 1A;

FIG. 2A is an electropherogram of a full scale reaction in accordancewith the present invention;

FIG. 2B is a raw trace of the reduced dye blobs of FIG. 2A;

FIG. 3A is an electropherogram of a half scale reaction in accordancewith the prior art;

FIG. 3B is raw trace of the reaction dye blobs of FIG. 3A;

FIG. 4A is an electropherogram of a half scale reaction in accordancewith the present invention;

FIG. 4B is a raw trace showing the absence of dye blobs of FIG. 4A;

FIG. 5A is an electropherogram of a quarter scale reaction in accordancewith the prior art;

FIG. 5B is raw trace showing the reaction dye blobs in FIG. 5A;

FIG. 6A is an electropherogram of a quarter scale reaction in accordancewith the present invention;

FIG. 6B is a raw trace showing the absence of dye blobs of FIG. 6A.

DETAILED DESCRIPTION

The present inventors have found that effective amounts of guanidine inthe sequencing reaction product disrupt aggregates and/or prevent theirformation, as evidenced by electropherograms even at higher reactionscales. Effective amounts of guanidine are amounts sufficient to reducethe presence of unincorporated dye terminators to an extent where theydo not deleteriously interfere with downstream analysis, particularlythe electrophoresis of the sequencing reaction products. Deleteriousinterference with electrophoresis is manifested in the appearance of dyeblobs in electropherograms, wherein the presence of the dye blobs makeit difficult or impossible to accurately resolve the sequencingproducts. Such interference is particularly noticeable with shorter DNA.Higher concentrations of dye terminators also interfere furtherdownstream during sequencing.

Excessive amounts of guanidine can result in the unacceptable loss ofshorter sequencing product. Insufficient amounts of guanidine can resultin inadequate reduction of deleterious artifacts, or the reappearancethereof. Suitable amounts of guanidine range from about 1 to about 60mM, more preferably from about 1 to about 30 mM, with about 5 to about10 mM being particularly preferred. It is surprising that onlyrelatively low concentrations of guanidine were effective insufficiently reducing or eliminating dye blobs from theelectropherograms.

Preferably the guanidine is used in the form of its salt. Suitable saltsinclude chaotropes of guanidine, such as guanidine carbonate orguanidine hydrochloride. The particular salt used should be chosen toavoid undesirable interactions in the reaction. Preferably the guanidinesalt is used in a low ionic solution, such as 0.3 mM EDTA of pH fromabout 5-11, preferably 8-10.

In its method aspects, the present invention comprises the steps ofproviding a defined quantity of sequencing reaction product, providingat least one ultrafiltration membrane having at least one surface,introducing the sequencing reaction product onto the surface of theultrafiltration membrane, adding the guanidine wash solution eitherprior to or after the sequencing reaction product is introduced onto theultrafiltration membrane surface, applying a driving force, such asconstant pressure differential or vacuum, to the surface of the membranesufficient to produce the sequencing reaction product substantially freeof contaminants. The addition of the guanidine wash solution andapplication of a driving force may be repeated. The sequencing reactionproduct is retained on the membrane surface while contaminants,including dye terminators, are filtered to waste. Preferably thesequencing reaction product is filtered to dryness (e.g., no visiblefluid remains), and the driving force is maintained for about 15 secondsafter filtration to dryness (where multiple filtrations are carried outsimultaneously in a multiwell device, preferably the driving force ismaintained until the last well is empty). The sequencing reactionproduct may be washed (such as with the wash solution of the presentinvention), filtered to dryness again, resuspended in a low ionicsolution known to those skilled in the art, such as formamide or water,and transferred (e.g., by pipetting) to an appropriate substrate forsequencing (e.g., electrokinetic injection), restriction digestion orfurther preparation.

The following Table illustrates suitable amounts of components forvarious concentrations of sequencing chemistries using the ABI BigDye®Terminator kit:

Reaction Strength Component 1x ½ x ½ x ¼ x ¼ x Template 2.0 μL 2.0 μL2.0 μL 2.0 μL 2.0 μL Plasmid (50 ng/μL) PCR (5 fmol/μL) 2.5x buffer* 0μL 4.0 μL 0 μL 2.0 μL 0 μL Primer (5 pmol/μL) 1.0 μL 1.0 μL 1.0 μL 1.0μL 1.0 μL BDT Premix 8.0 μL 4.0 μL 4.0 μL 2.0 μL 2.0 μL Milli-Q ™ H₂O9.0 μL 9.0 μL 3.0 μL 3.0 μL 0 μL Total 20 μL 20 μL 10 μL 10 μL 5 μL*2.5x Buffer: 200 mM Tris-HCl, pH 9.0, 5 mM MgCl₂; BigDye mix, primer,2.5x buffer and water can be mixed together to make sequencing“cocktail”; an appropriate volume of cocktail is then dispensed intoeach well.

Suitable ultrafiltration membranes have molecular cutoffs between about1000 and 30,000 Daltons, preferably between about 3,000 and 15,000Daltons. They may be made from a variety of materials, including but notlimited to polyamides, polysulphones, polyethersulphones,polyarylsulphones, cellulosics, regenerated cellulose and polyvinylidenefluoride. They may be symmetrical or asymmetrical, with the latter beingpreferred. For high throughput applications, preferably a plurality ofultrafiltrations are carried out simultaneously, most preferably using a384 well filter plate. Other size plates, including 96 well format, alsocan be used. Suitable filter plates include the MultiScreen® or MONTAGE™384-SEQ filter plates commercially available from Millipore Corporation.

Suitable vacuum is that sufficient to reduce or remove the dyeterminators, and generally ranges from about 15 to about 28 inches ofmercury, preferably from about 23-25 inches of mercury. Generally thedriving force is applied slightly (about 15 seconds) longer than thetime necessary to remove all visible liquid from the wells, whichtypically is about 3-4 minutes.

EXAMPLE 1

A sequencing reaction was set up in a thermal cycling plate (96 wells)and amplified using an appropriate thermal cycling program. The reactioncomponents were 5 pmol of M13 primer, 200 ng of pLH2 (template), 8 μl ofBigDye pre-mix, and sufficient Milli-Q® water to bring the final volumeto 20 μl. All of the mixture was transferred to a Montáge SEQ₉₆ platecommercially available from Millipore Corporation. The plate was placedon a vacuum manifold and vacuum of 23-25 inches Hg was applied for 3-4minutes until no visible fluid remained in the wells. Vacuum wascontinued for about 15 seconds, and the plate was removed from themanifold. Excess fluid was removed from the bottom of the plate bypressing the plate briefly to a stack of paper towels. The plate wasreturned to the manifold, and 25 μl of was solution (no guanidine) wasadded. Vacuum was again applied at 23-25 inches Hg for 3-4 minutes untilno visible fluid remained in the wells, and for about 15 secondsthereafter. The plate was removed from the manifold and excess fluidagain was removed from the bottom of the plate by pressing the plateagainst a stack of paper towels. 25 μl of injection solution was added,and the DNA was resuspended by pipetting up and down 15-20 times. Thepurified sequencing products were injected into an ABI 3700 sequencer at2 KV, 15 seconds. The result is shown in FIGS. 1A and 1B. Large dyeblobs are apparent.

EXAMPLE 2

A sequencing reaction was set up in a thermal cycling plate (96 wells)and amplified using an appropriate thermal cycling program. The reactioncomponents were 5 pmol of M13 primer, 200 ng of pLH2 (template), 8 μl ofBigDye pre-mix, and sufficient Milli-Q® water to bring the final volumeto 20 μl. 30 μl of wash solution (no guanidine) was added to eachsequencing reaction. After thermal cycling, 30 μl of sequencing washsolution comprising 0.5 mM guanidine in 0.3 mM EDTA of pH 8 was added toeach of the reactions and mixed gently. All of the mixture wastransferred to a Montáge SEQ₉₆ plate commercially available fromMillipore Corporation. The plate was placed on a vacuum manifold andvacuum of 23-25 inches Hg was applied for 3-4 minutes until no visiblefluid remained in the wells. Vacuum was continued for about 15 seconds,and the plate was removed from the manifold. Excess fluid was removedfrom the bottom of the plate by pressing the plate briefly to a stack ofpaper towels. The plate was returned to the manifold, and 25 μl of thesequencing wash solution was added. Vacuum was again applied at 23-25inches Hg for 3-4 minutes until no visible fluid remained in the wells,and for about 15 seconds thereafter. The plate was removed from themanifold and excess fluid again was removed from the bottom of the plateby pressing the plate against a stack of paper towels. 25 μl ofinjection solution was added, and the DNA was resuspended by pipettingup and down 15-20 times. The purified sequencing products were injectedinto an ABI 3700 sequencer at 2 kV, 15 seconds. The result is shown inFIGS. 2A and 2B. The reduction in dye blobs compared to FIGS. 1A and 1Bis apparent.

EXAMPLE 3

The same procedure as in Example 1 was carried out except at halfreaction scale, wherein only 4 μl of BigDye pre-mix was used, and thefinal volume was 10 μl. The results are shown in FIGS. 3A and 3B.

EXAMPLE 4

The same procedure as in Example 2 was carried out except at halfreaction scale, wherein only 4 μl of BigDye pre-mix was used, and thefinal volume was 10 μl. The results are shown in FIGS. 4A and 4B. Theabsence of dye blobs is apparent in comparison to FIGS. 3A and 3B.

EXAMPLE 5

The same procedure as in Example 1 was carried out except at quarterreaction scale, wherein only 2 μl of BigDye pre-mix was used, and thefinal volume was 5 μl. The injection into an ABI 3100 Avant sequencerwas at 1 kV, 22 seconds. The results are shown in FIGS. 5A and 5B.

EXAMPLE 6

The same procedure as in Example 2 was carried out except at halfreaction scale, wherein only 2 μl of BigDye pre-mix was used, and thefinal volume was 5 μl. The injection into an ABI 3100 Avant sequencerwas at 1 kV, 22 seconds. The results are shown in FIGS. 6A and 6B. Theabsence of dye blobs is apparent in comparison to FIGS. 5A and 5B.

1. A method for purifying sequencing reaction product by removingunincorporated dye terminators from a sequencing reaction, comprising:a) providing sequencing reaction product having unincorporated dyeterminators; b) providing at least one ultrafiltration membrane havingat least one surface; c) providing a solution comprising an amount ofguanidine less than 5 mM effective for removing the unincorporated dyeterminators from said sequencing reaction; d) introducing saidsequencing reaction product and said solution to said at least onesurface of said ultrafiltration membrane; and e) applying a drivingforce to said ultrafiltration membrane to produce purified sequencingreaction product by removing unincorporated dye terminators from thesequencing reaction product.
 2. The method of claim 1, furthercomprising resuspending said purified sequencing reaction product in alow ionic solution.
 3. The method of claim 2, further comprisingtransferring said resuspended sequencing reaction product to a substratefor sequencing.
 4. The method of claim 1, wherein said solutioncomprising said amount of guanidine further comprises EDTA.
 5. Themethod of claim 1, wherein said amount of guanidine is from about 0.5 mMto less than 5 mM.
 6. The method of claim 1, wherein said amount ofguanidine is from about 0.5 mM to about 1 mM.
 7. The method of claim 1,wherein in step (e) the driving force is maintained until the purifiedsequencing reaction product is filtered to dryness such that no visiblefluid remains.
 8. The method of claim 1, wherein in step (d) theguanidine solution is added onto the ultrafiltration membrane surfaceprior to the addition of the sequencing reaction product onto theultrafiltration membrane surface.
 9. The method of claim 1, wherein instep (d) the guanidine solution is added onto the ultrafiltrationmembrane surface after to the addition of the sequencing reactionproduct onto the ultrafiltration membrane surface.
 10. The method ofclaim 2, wherein the low ionic solution is selected from the groupconsisting of water, formamide and mixtures thereof.
 11. The method ofclaim 1, wherein in step (d) the ultrafiltration membrane has amolecular cutoff between about 1,000 and 30,000 Daltons.
 12. The methodof claim 1, wherein in step (d) the ultrafiltration membrane has amolecular cutoff between about 3,000 and 15,000 Daltons.
 13. The methodof claim 1, wherein in step (d) the ultrafiltration membrane comprises amaterial selected from the group consisting of polyamides,polysulphones, polyethersulphones, polyarylsulphones, cellulosics,regenerated cellulose, polyvinylidene fluoride and combinations thereof.14. The method of claim 1, wherein in step (c) guanidine comprisesguanidine hydrochloride.
 15. The method of claim 1, wherein in step (c)guanidine comprises guanidine carbonate.
 16. A method of removingunincorporated dye terminators from an unpurified DNA sequencingreaction product, comprising: a) providing at least one ultrafiltrationmembrane having at least one surface; b) providing a wash solutioncomprising EDTA and an amount of guanidine or salt thereof from about0.5 to less than 5 mM effective for removing unincorporated dyeterminators from the unpurified DNA sequencing reaction product; c)introducing the wash solution to the surface of the ultrafiltrationmembrane; d) providing the unpurified DNA sequencing reaction producthaving unincorporated dye terminators; e) introducing the unpurified DNAsequencing reaction product having the unincorporated dye terminators tothe wash solution located on the surface of the ultrafiltrationmembrane; and f) applying a driving force to the ultrafiltrationmembrane to remove unincorporated dye terminators from the DNAsequencing reaction product, producing a purified DNA sequencingreaction product.
 17. The method of claim 16, wherein in step (f) thedriving force is maintained until the purified sequencing reactionproduct is filtered to dryness such that no visible fluid remains. 18.The method of claim 16, further comprising resuspending said purifiedDNA sequencing reaction product in a low ionic solution selected fromthe group consisting of water, formamide and mixtures thereof.
 19. Themethod of claim 18, further comprising transferring said resuspendedsequencing reaction product to a substrate for sequencing.
 20. Themethod of claim 16, wherein in step (d) the ultrafiltration membrane hasa molecular cutoff between about 1,000 and 30,000 Daltons.
 21. Themethod of claim 16, wherein in step (c) guanidine comprises guanidinehydrochloride.
 22. The method of claim 16, wherein in step (c) guanidinecomprises guanidine carbonate.
 23. A method of removing unincorporateddye terminators from an unpurified DNA sequencing reaction product,comprising: a) providing an unpurified DNA sequencing reaction producthaving unincorporated dye terminators; b) providing at least oneultrafiltration membrane having at least one surface; c) introducing theunpurified DNA sequencing reaction product having unincorporated dyeterminators to the surface of the ultrafiltration membrane; d) providinga wash solution comprising EDTA and an amount of guanidine or saltthereof from about 0.5 mM to less than 5 mM effective for removing theunincorporated dye terminators from the unpurified DNA sequencingreaction product; e) introducing the wash solution to the unpurified DNAsequencing reaction product having unincorporated dye terminatorslocated on the surface of the ultrafiltration membrane; and f) applyinga driving force to the ultrafiltration membrane to remove theunincorporated dye terminators from the DNA sequencing reaction product,producing a purified DNA sequencing reaction product.
 24. The method ofclaim 23, wherein in step (d) guanidine comprises guanidinehydrochloride.
 25. The method of claim 23, wherein in step (c) theultrafiltration membrane has a molecular cutoff between about 3,000 and15,000 Daltons.
 26. The method of claim 23, further comprisingresuspending said purified sequencing reaction product in a low ionicsolution, and transferring said resuspended sequencing reaction productto a substrate for sequencing.
 27. The method of claim 23, wherein saidsolution comprising said amount of guanidine further comprises EDTA. 28.The method of claim 23, wherein in step (f) the driving force ismaintained until the purified sequencing reaction product is filtered todryness such that no visible fluid remains.
 29. The method of claim 23,wherein in step (c) the ultrafiltration membrane comprises a well filterplate.